Cloning of the Major Outer Membrane Protein Expression Locus in Anaplasma platys and Seroreactivity of a Species-Specific Antigen

Abstract

Anaplasma platys infects peripheral blood platelets and causes infectious cyclic thrombocytopenia in canines. The genes, proteins, and antigens of A. platys are largely unknown, and an antigen for serodiagnosis of A. platys has not yet been identified. In this study, we cloned the A. platys major outer membrane protein cluster, including the P44/Msp2 expression locus (p44ES/msp2ES) and outer membrane protein (OMP), using DNA isolated from the blood of four naturally infected dogs from Venezuela and Taiwan, Republic of China. A. platys p44ES is located within a 4-kb genomic region downstream from a putative transcriptional regulator, tr1, and a homolog of the Anaplasma phagocytophilum, identified here as A. platys omp-1X. The predicted molecular masses of the four mature A. platys P44ES proteins ranged from 43.3 to 43.5 kDa. Comparative analyses of the deduced amino acid sequences of Tr1, OMP-1X, and P44/Msp2 proteins from A. platys with those from A. phagocytophilum showed sequence identities of 86.4% for Tr1, 45.9% to 46.3% for OMP-1X, and 55.0% to 56.9% for P44/Msp2. Comparison between A. platys and Anaplasma marginale proteins showed sequence identities of 73.1% for Tr1/Tr, 39.8% for OMP-1X/OMP1, and 41.5% to 42.1% for P44/Msp2. A synthetic OMP-1X peptide was shown to react with A. platys-positive sera but not with A. platys-negative sera or A. phagocytophilum-positive sera. Together, determination of the genomic locus of A. platys outer membrane proteins not only contributes to the fundamental understanding of this enigmatic pathogen but also helps in developing A. platys-specific PCR and serodiagnosis.

INTRODUCTION

Anaplasma platys (formerly known as Ehrlichia platys) was first identified in 1978 in Florida as a Rickettsia-like bacterium found in the platelets of dogs with infectious canine cyclic thrombocytopenia (ICCT) (26). The original description noted the morphological and biological similarity of this bacterium to Ehrlichia canis in infected dogs and to Anaplasma marginale in infected cattle, both of which are current members of the family Anaplasmataceae (17, 26). Indirect fluorescent antibody testing of platelet-rich plasma from dogs experimentally infected with ICCT showed minimal serological cross-reactivity between the new bacterial species and E. canis; therefore, a new bacterial species was proposed and named “Ehrlichia platys” (23). In 1992, Anderson et al. (3) reported the 16S rRNA gene sequence of A. platys in Louisiana. Subsequently, the groEL gene sequence of A. platys was also determined (29, 67). Phylogenetic analysis of these two gene sequences showed that the new bacterial species was distinct from but closely related to Anaplasma phagocytophilum and A. marginale, which led to reclassification of the new bacterial species into the genus Anaplasma (17). A. platys inclusions in the platelets from a naturally infected dog were shown to cross-react with mouse anti-A. phagocytophilum serum (32).

Clinical signs of ICCT are fever, depression, and anorexia (23). Parasitemia and thrombocytopenia occur in cycles of approximately 10 to 14 days (23). In the United States, dogs that are seropositive for A. platys have been found in Florida, Louisiana, Mississippi, Texas, Arkansas, North Carolina, Pennsylvania, Illinois, Idaho, and California, and dogs were frequently seropositive for both A. platys and E. canis (23). Internationally, A. platys DNA has been detected in blood of dogs from Brazil (21), Greece (43), France (33), Spain (54), Portugal (13), Taiwan (15), China (28), Japan (61), Thailand and Venezuela (29, 59), Australia (12), and the Republic of the Congo (55). A. platys has been found in the brown dog tick Rhipicephalus sanguineus in Japan (34), Spain (58), and the Republic of the Congo (55); however, it has not been proven whether R. sanguineus is a biological vector of A. platys (56). A. platys has not been isolated in culture, and the genes, proteins, and antigens of A. platys are not known.

In A. phagocytophilum and A. marginale, surface-exposed, immunodominant, 44-kDa major outer membrane proteins (OMPs) (P44s/Msp2s) are encoded by the p44/msp2 polymorphic multigene family (6, 9, 39, 41, 69–71). In A. phagocytophilum, P44 proteins consist of a single central hypervariable region of approximately 94 amino acids (aa) and N-terminal and C-terminal conserved regions of approximately 186 and 146 amino acids, respectively (41). A single polymorphic p44/msp2 expression locus (p44/msp2ES) is found in the genome of A. phagocytophilum and A. marginale (10, 18). In both species, this expression locus is found downstream from the tr1/tr gene, which encodes a putative transcription factor, and homologs of Ehrlichia omp-1/p28/p30/map-1 genes, which encode immunodominant polymorphic major OMPs (6, 8, 39). At the p44/msp2ES locus, p44s and msp2 donor sequences from elsewhere in the genome undergo gene conversion via a RecF-dependent pathway, allowing the expression of various p44 donor sequences at this locus via a single promoter (6, 8, 39, 40). This mechanism is thought to facilitate P44/Msp2 antigenic variation during acute and persistent infection and to facilitate adaptation to new environments, such as during transmission between tick and mammalian hosts (7, 11, 38, 40, 65, 71). Purified native P44 from A. phagocytophilum and purified native OMP-1s (P28 and OMP-1F) of Ehrlichia chaffeensis have porin activity (30, 37).

In the present study, we first isolated a major outer membrane protein expression locus in A. platys. Second, we compared the major outer membrane protein expression loci among A. platys, A. marginale, and A. phagocytophilum. Third, we analyzed the structure of major outer membrane proteins of A. platys using bioinformatics tools. Fourth, we determined A. platys-specific amino acid sequences predicted to be antigenic and located in the external loop regions of β-barrel proteins. Finally, we tested the immunoreactivity of one peptide by enzyme-linked immunosorbent assay (ELISA) using A. platys-positive canine serum. The results suggest the potential to use these peptides for serodiagnosis of A. platys infection.

MATERIALS AND METHODS

A. platys-infected dogs.

Dogs that were naturally infected with A. platys were identified in Lara, Venezuela, in 2007 by observation of bacterial inclusions (morulae) in platelets from blood smears, and cases were confirmed by PCR and sequencing using primer pairs specific for A. platys 16S rRNA (EP1-EP3 and EP2-EP3) (29). Naturally infected dogs in Taichung, Taiwan, Republic of China, were identified in 2010 and confirmed by PCR using the primer pair EPLAT5-EPLAT3 (42).

Cloning of p44 expression locus from A. platys.

DNA samples from three dogs from Venezuela and one dog from Taiwan were used as templates. By aligning the p44/msp2 expression loci from A. phagocytophilum and A. marginale, we were able to design several degenerate primers for conserved regions of the locus (Fig. 1; primers are available upon request). Using the first and the second primer pairs, F1-R1 and F1-R2, (hemi-)nested touchdown PCR (52) was used to amplify the tr1 and omp-1X gene sequences from A. platys. In order to avoid truncating p44 pseudogenes in the A. platys genome, we designed primer F3 upstream of the predicted p44 open reading frame. p44ES sequences were amplified by nested touchdown PCR using primer pairs F2-R3 and F3-R4. Amplification was performed as previously described (68). The amplified DNA fragments were cloned using a TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced with M13 forward or M13 reverse sequencing primers. All sequencing data were assembled using the SeqMan program (DNASTAR, Inc., Madison, WI). To confirm the assembly, the entire locus was amplified using primers F1 and R5 (primers are available upon request).

Fig. 1.

Strategy for A. platys (A. pl) major outer membrane protein expression locus sequencing. (A) A. phagocytophilum (A. ph) p44ES and A. marginale (A. ma) msp2ES were aligned to design primer F1 (targeting the highly conserved region upstream of tr1/tr) and the degenerate primers R1 (targeting the p44ES/msp2 C-terminal region), R2 (targeting the conserved intergenic region between omp-1X/omp1 and omp-1N/opag3), R3 (targeting the p44ES/msp2 N-terminal region), and R4 (targeting the conserved valS gene downstream of p44ES/msp2ES). Primers F2 and F3, indicated in panel B, were designed based on the sequence results. (B) The final sequence (3,957 bp) was assembled using the SeqMan program within the DNASTAR software. Genes are represented as boxes with arrows indicating their orientation. Numbers indicate base pairs. (C) The entire expression locus fragment D (arrowhead) amplified from the dog 2 blood DNA specimen by primers F1 and R5.

Phylogenetic analysis.

The deduced amino acid sequences for Tr1, OMP-1/OMP-1X, and P44ES from A. marginale, A. phagocytophilum, and A. platys were aligned using the MegAlign program (DNASTAR, Inc.) by the Clustal W method.

Protein structure analysis using bioinformatics tools.

The SignalP 3.0 server trained on Gram-negative bacteria (http://www.cbs.dtu.dk/services/SignalP/) was used for signal peptide sequence analysis. The secondary structures of P44 and OMP-1X were predicted by PRED-TMBB (4) and hydrophobicity analysis and the hydrophobic moment profile method, as previously described (30, 35). The antigenic index and surface probability were determined using the Protean program (DNASTAR, Inc.).

ELISA analysis of OMP-1X-specific peptide.

The OMP-1X peptide from A. platys was synthesized at Biomatik (Wilmington, DE). The purity of the peptide was greater than 98%, as assessed by high-performance liquid chromatography. The wells of a 96-well microtiter plate were coated with 200 ng peptide/well, and the ELISA was performed as previously described (60). Samples were from three dogs that were PCR positive for A. platys (TW 431, TW 270, and TW 210) and three dogs that were both PCR negative for A. platys and antigen dot blot negative for A. phagocytophilum (E05-290, E10-0062, and E10-0075). In addition, horse anti-A. phagocytophilum-positive sera (EQ002, EQ006, and E09-0011) (65, 71) were used to confirm the absence of OMP-1X peptide antigen cross-reactivity with anti-A. phagocytophilum antibodies. The horseradish peroxidase substrate 2,2′-azido-di-(3-ethyl)-benzthiazoline-6-sulfonic acid (Sigma, St. Louis, MO) in 70 mM citrate buffer (pH 4.2) was applied, and absorbance values at 415 and 492 nm were measured in an ELISA plate reader (Molecular Devices, Sunnyvale, CA) as previously described (68). The results are presented as the optical density at 415 nm minus that at 492 nm (OD₄₁₅ − OD₄₉₂), and the cutoff for a positive reaction was set at greater than the mean of the value for the formula OD₄₁₅ − OD₄₉₂ + 3 standard deviations (SD) for the negative-control samples (OD > 0.165). The assay was repeated at least three times.

Nucleotide sequence accession numbers.

The A. platys tr1-omp-1X-p44ES sequences from two naturally infected dogs from Venezuela were assembled and deposited at GenBank under accession numbers GQ868750 and GU357491. Additional p44ES and p44 sequences were deposited at GenBank under accession numbers GU357492, GU357493, GU357494, GU357495, GU357496, GU357497, and HQ738571.

RESULTS

Cloning of the A. platys outer membrane protein expression locus.

We designed three degenerate primers and one primer at the highly conserved upstream region of tr1 based on alignment of the tr1-omp1X-omp1N-p44 gene cluster from A. phagocytophilum (GenBank sequence accession number AY137510) with the tr-omp1-opag3-opag2-opag1-msp2 gene cluster from A. marginale (GenBank sequence accession number AY132308) (Fig. 1, primers are available upon request). The first touchdown PCR (52) was designed to amplify the entire cluster fragment using primers F1 and R1 (Fig. 1) (primers are available upon request). The PCR products were then used as templates for heminested touchdown PCR using primers F1 and R2 (Fig. 1) (primers are available upon request). A single band, approximately 2,100 bp in size, was amplified (fragment A). The PCR product was cloned using a TA cloning kit and then sequenced. The results showed that fragment A contained A. platys tr1 and a homolog of omp-1X (named here A. platys omp-1X).

The region downstream from fragment A was amplified by nested touchdown PCR using the PCR products obtained with primers F1 and R1 as a template, with primer F2 based on fragment A and primer R3 based on conserved sequences for p44 (msp2) in A. phagocytophilum and A. marginale. A single band, approximately 1,100 bp in size, was amplified (fragment B). The PCR product was cloned using the TA cloning kit, and sequencing showed that fragment B contained a partial sequence for A. platys p44ES. To amplify the full-length p44ES from A. platys, primer F3 was designed based on fragment B and primer R4 was designed based on the conserved region of valS found downstream from p44 (msp2) in A. phagocytophilum and A. marginale. Another touchdown PCR was conducted, using primers F1 and R4. The PCR products were then used as templates for heminested touchdown PCR using primers F3 and R4. A single band, approximately 1,700 bp in size, was amplified (fragment C). The PCR products were cloned using the TA cloning kit, and sequencing showed that fragment C contained the full-length p44 sequence from A. platys.

The final assembled sequence of 3,957 bp from Venezuelan dogs 1 and 2 contained the entire A. platys p44ES locus. The average sequence coverage of the entire locus is 8.3-fold (5- to 15-fold). To confirm that the assembly is from a complete genomic locus, we designed primer R5 downstream of the predicted p44 open reading frame and conducted one more touchdown PCR using primers F1 and R5. A single band of approximately 3.9 kb was amplified (fragment D), indicating that the fragment containing the entire locus was amplified from the blood of dog 2 (Fig. 1C). The G+C content was determined to be 47.46% to 47.51%. The synteny among the entire outer membrane protein gene clusters of A. platys and the two previously sequenced Anaplasma species was analyzed using the Artemis Comparison Tool (14). The tr1 sequences were conserved among the three Anaplasma species (Fig. 2). The 5′ and 3′ regions of p44ES were conserved between A. platys and A. phagocytophilum but less conserved between A. platys and A. marginale (Fig. 2).

Fig. 2.

Synteny analysis, using the Artemis comparison tool, of the A. platys (A. pl) p44ES cluster relative to those of A. phagocytophilum (A. ph) and A. marginale (A. ma). Each diagonal bar corresponds to a good match. Numbers indicate base pairs. Score cutoff, 140.

A. platys Tr1 structure.

Three similar (97.8% to 99.5%) A. platys tr1 sequences were obtained from two dogs from Venezuela and one dog from Taiwan. The predicted molecular mass of A. platys Tr1 was 21.0 to 21.1 kDa, and the isoelectric point was 5.50 to 5.80 (Table 1). Tr1 was not predicted to have a signal peptide and, thus, is a cytoplasmic protein, as analyzed by SignalP 3.0. Tr1 was predicted to contain a putative N-terminal helix-turn-helix DNA-binding domain, based on the analysis of the NCBI conserved domain database, suggesting that it is a transcriptional regulator. The amino acid sequence identity between A. platys Tr1 and A. phagocytophilum Tr1 (GenBank sequence accession number YP_505749) was 84.8% to 86.4%, and that between A. platys Tr1 and A. marginale Tr (GenBank sequence accession number YP_154239) was 73.1% to 74.1%.

Table 1.

Properties of the A. platys p44ES cluster

Protein	Upstream intergenic space (bp)	Gene length (bp)	Protein size (amino acids)a	Signal peptide (amino acids)b	Molecular mass (Da)a,d	Isoelectric pointd
Tr1	NAc	558	185	NA	21010.6–20952.6	5.50–5.80
OMP-1X	306	933	301	23	31885.0–31942.1	7.27–7.92
P44ES	682	1,221	380–386d	21	41167.3–41359.5	5.30–5.72

^aMature protein.

^bPredicted cleavage site.

^cNA, not applicable.

^dRange among strains or alleles.

A. platys OMP-1X structure.

Three nearly identical (99.1%) A. platys omp-1X sequences were obtained from two dogs from Venezuela and one dog from Taiwan. Using the SignalP 3.0 server, OMP-1X was predicted to have a signal peptide with a cleavage site between positions 23 and 24. The predicted molecular mass of mature A. platys OMP-1X was 31.9 kDa and the isoelectric point 7.27 to 7.92 (Table 1). We then examined the secondary structure of OMP-1X, using PRED-TMBB (4). The discrimination value of the OMP-1X amino acid sequence was 2.907, which is below the threshold value of 2.965, making OMP-1X likely to be a β-barrel protein localized to the outer membrane. Hydrophobicity analysis and the hydrophobic moment profile program, developed for porin structure prediction (35), predicted 14 β-strands in OMP-1X. The protein sequences most closely related to A. platys OMP-1X are A. phagocytophilum OMP-1X (GenBank sequence accession number YP_505750; 45.9%-46.3% identity) and A. marginale OMP-1 (GenBank sequence accession number YP_154240; 39.8% identity). A phylogenetic analysis showed that OMP-1X homologs in Anaplasma spp. form a cluster that is distinct from the cluster of most closely related OMP-1X homologs in each Ehrlichia spp. (Fig. 3).

Fig. 3.

Phylogram of A. platys (A. pl) OMP-1X protein and related proteins from A. phagocytophilum (A. ph), A. marginale (A. ma), E. canis (E.ca), E. chaffeensis (E.ch), E. ewingii (E. ew), and E. ruminantium (E. ru). The tree was constructed using the MegAlign Clustal W method within the DNASTAR software. GenBank sequence accession numbers are shown in parentheses.

A. platys P44ES structure.

Four P44ES sequences (GenBank sequence accession numbers GQ868750, GU357491, GU357492, and GU357493) were obtained from three dogs from Venezuela. Using the SignalP 3.0 server, P44ES was predicted to have a putative signal peptide with a cleavage site between positions 21 and 22. The molecular mass of the mature P44ES protein was predicted to be 41.2 to 41.4 kDa and the isoelectric point 5.30 to 5.72 (Table 1). By PRED-TMBB (4) analysis, the discrimination value of the P44 amino acid sequence was 2.920, which is below the threshold value of 2.965, making P44 likely to be a β-barrel protein localized to the outer membrane. Hydrophobicity analysis and the hydrophobic moment profile predicted 16 β-strands in P44. Alignment of a total of nine A. platys P44 sequences (the four P44 full-length proteins from dogs 1, 2, and 3 and the five partial P44 sequences obtained from dogs 1, 2, and Taiwan) using the HVF and HVR primers revealed a single central hypervariable region (aa position 193 to 247) of approximately 55 amino acid residues and N-terminal and C-terminal conserved regions of approximately 192 and 159 amino acid residues, respectively. The conserved amino acids C, C, W, and A from the P44 hypervariable region of A. phagocytophilum P44 (41) were also detected in the hypervariable region of A. platys P44. The C terminus of A. platys P44 ends with phenylalanine, as does the C terminus of A. phagocytophilum P44 (30). The amino acid sequence identity between A. platys P44ES and A. phagocytophilum P44-18ES (GenBank sequence accession number YP_505752) was 55.0% to 56.9%, and that between A. platys P44ES and A. marginale Msp2 (GenBank sequence accession number YP_154245) was 41.5% to 42.1%. Phylogenetic analysis placed full-length A. platys p44s between A. phagocytophilum p44s and A. marginale msp2s (Fig. 4). The sequence identities of the conserved N-terminal 192 amino acids and the conserved C-terminal 159 amino acids of A. platys and A. phagocytophilum P44s were 57.3% and 66.7%, respectively.

Fig. 4.

Phylogram of P44ES/Msp2 proteins, indicated by their GenBank accession numbers, of A. platys (A. pl), A. phagocytophilum (A. ph), and A. marginale (A. ma). A total of 23 P44/Msp2 proteins were segregated into four clusters. The tree was constructed using the MegAlign Clustal W method within the DNASTAR software.

Primer pairs (HVF and HVR; primers are available upon request) designed based on the A. platys p44 conserved region amplified only A. platys DNA and not A. phagocytophilum and A. marginale DNA (data not shown). Alignment of a total of nine A. platys P44 hypervariable regions and flanking conserved regions with P44/Msp2 sequences among A. phagocytophilum P44s and A. marginale Msp2s revealed several A. platys-specific sequences: TGTAAGSDVDYVSKF (aa position 23 to 37), TRVEWKAE (aa position 78 to 85), AAEIVKFAEAVGTSAK (aa position 174 to 189), SWKCTQTG (aa position 207 to 214), AAKAEDLS (aa position 248 to 255), and ATTNKTKEF (aa position 378 to 386). These A. platys-specific p44 regions could be utilized as serologic test antigens to distinguish A. platys infections from A. phagocytophilum or A. marginale infections.

ELISA analysis of OMP-1X.

When the Clustal W method was used to compare A. platys OMP-1X to its phylogenetically closest OMP-1 homologs—A. phagocytophilum OMP-1X (YP_505750), A. marginale OMP1 (YP_154240), E. canis P30-19 (AAK28680), Ehrlichia ruminantium Map1-related protein (YP_180721), Ehrlichia ewingii OMP-1-1 (ABO36240), and E. chaffeensis OMP-1 M (YP_507903) (GenBank sequence accession numbers are in parentheses)—we identified a unique region in the A. platys OMP-1X amino acid sequence. This sequence, AVQEKKPPEA, is within the 2nd external loop from the N terminus based on the hydrophobicity analysis and the hydrophobic moment profile program. The sequence is predicted by the Protean program to be highly antigenic and surface exposed, which may aid in differential serodiagnosis (Fig. 5 A). The A. platys OMP-1X peptide was synthesized, and its reactivity to known infected dog sera was tested by ELISA. Three A. platys PCR-positive dog sera reacted with the synthesized OMP-1X peptide antigen. Sera from A. platys PCR-negative dogs and horse anti-A. phagocytophilum serum did not react with the OMP-1X peptide antigen (Fig. 5B), suggesting that this antigen can be used for species-specific serodiagnosis of A. platys.

Fig. 5.

(A) The shaded graphs show antigenic index and surface porbability profiles of the A. platys OMP-1X fragment (aa positions 50 to 90) determined (by Jameson-Wolf algorithm and Emini analyses, respectively) using the Protean program of DNASTAR. The y axis shows calcuated values. Alignment of the A. platys (A. pl) OMP-1X protein fragment (aa positions 50 to 90) with related proteins, indicated by their GenBank accession numbers, from A. phagocytophilum (A. ph), A. marginale (A. ma), E. canis (E. ca), E. chaffeensis (E. ch), E. ewingii (E. ew), and E. ruminantium (E. ru) using the Clustal W method revealed residues that are conserved (solid-line boxes) and a unique region in A. platys OMP-1X (AVQEKKPPEA; dashed-line box). This region is specific to A. platys and predicted to be antigenic and surface exposed. (B) The A. platys-specific peptide was synthesized and tested by ELISA for reactivity to sera from A. platys PCR-positive dogs (bars 1 to 3), A. platys PCR-negative dogs (bars 4 to 6), and A. phagocytophilum-seropositive horses (bars 7 to 9). The y axis shows values for (OD₄₁₅ − OD₄₉₂) ± SD. A reaction was considered positive when the value was greater than the mean value for (OD₄₁₅ − OD₄₉₂) + 3 SD for negative-control plasma (dashed line). The data shown are representative of triplicate assays.

DISCUSSION

In the present study, the entire 4-kb A. platys major outer membrane protein expression locus, containing the tr1, omp-1X, and p44 genes, was sequenced, providing new insight into the p44 expression locus and major surface antigens of A. platys. Different p44ES sequences were detected from individual dogs infected with A. platys, suggesting that there is a mixed P44 allele population of A. platys, similar to observations for A. phagocytophilum p44 expression in humans, mice, and horses (38, 39, 65) and A. marginale msp2 expression in cattle (19, 22, 47). The p44 primer pair HVF and HVR, designed in this study, can be used to obtain a more complete A. platys repertoire of p44 sequences in various geographic regions in order to learn about P44 antigen diversity among A. platys strains. Future analysis should determine whether multiple copies of p44 are present in the A. platys genome, as is the case for A. phagocytophilum p44 and A. marginale msp2 (10, 18, 69); this would further contribute to understanding the p44/msp2 multigene family, which is characterized by highly active intragenomic recombination.

Our synteny analysis suggests that the major outer membrane expression locus existed in a common ancestor of the three Anaplasma species in existence today. Furthermore, the locus appears to have diverged primarily by duplicating omp-1-like sequences between tr1 and p44/msp2ES; A. marginale, A. phagocytophilum, and A. platys have 4, 2, and 1 omp-1-like sequences, respectively. The three species of Anaplasma infect different types of host cells, namely, erythrocytes, neutrophils, and platelets. Comparative studies of P44/Msp2s and OMP-1 homologs from A. marginale, A. phagocytophilum, and A. platys may provide a new approach to investigate the host cell tropism of Anaplasma spp.

Tr1, a putative transcription factor, is more highly expressed in tick cells infected with A. phagocytophilum than in human leukemic HL-60 cells infected with A. phagocytophilum, suggesting that Tr1 may regulate genes involved in the bacterial infection cycle in ticks (44, 64). In contrast, Tr is expressed similarly in bovine red blood cells and IDE8 tick cell cultures infected with A. marginale (5). Whether the expression of A. platys Tr1 differs in infected platelets and tick cells is unknown. In A. phagocytophilum tr1, two omp-1s and p44ES are coexpressed (39). In cattle blood, A. marginale tr, omp-1, opag1-3, and msp2 are coexpressed (48). OMP-1 homologous proteins are major surface antigens in Ehrlichia species (24, 46, 57, 62, 66), and OMP-1X may function similarly in the A. platys infection cycle. A. platys OMP-1X is predicted to have a β-barrel structure similar to those of E. chaffeensis P28 and OMP-1F (37) and is thus probably a porin.

In A. phagocytophilum and A. marginale, the P44/Msp2 transcript profiles in mammals and ticks are distinct, which may reflect an adaptation to physiological differences between these species (44, 53, 71). Furthermore, conversion of the A. phagocytophilum p44 gene within mammalian hosts suggests that P44 plays a role in antigenic variation (8, 20, 38, 65). In cattle, A. marginale Msp2 proteins provide the antigenic variation necessary for persistent infection (6, 11, 47). A. platys P44 is, therefore, expected to play an important role in determining persistent or cyclical rickettsemia. It is not known whether A. platys p44ES undergoes nonsegmental gene conversion (as in A. phagocytophilum to generate identical P44s from a large number of donor loci) or segmental gene conversion (as in A. marginale to generate mosaic Msp2ES from a small number of donor loci) (39, 47). P44 has a role in the interaction between A. phagocytophilum and host cells (36, 49, 64). P44 also has porin activity that allows for passive diffusion of hydrophilic solutes (30). A. platys P44 is predicted to have a β-barrel structure similar to that of A. phagocytophilum P44 and is thus probably a porin.

A. phagocytophilum is known to infect dogs in regions where the Ixodes tick is endemic (2, 25, 50, 51). A. platys inclusions in the platelets of a naturally infected dog cross-reacted with mouse anti-A. phagocytophilum serum (32). It is important, therefore, to develop a method for distinguishing A. platys infection from A. phagocytophilum infection. Since the p44 primer pair HVF and HVR, designed in the present study, is specific to A. platys, it is expected to be useful for species-specific PCR diagnosis. P44 of A. phagocytophilum is the major surface antigen used for serologic diagnosis of human granulocytic anaplasmosis (1, 16, 27, 31, 70). In the present study, we could identify several A. platys-specific amino acid sequences within P44 proteins that can be used as serologic test antigens to provide differential diagnosis from other Anaplasma species infections. Additionally, Ehrlichia OMP-1/P28/P30/MAP families are immunodominant major outer membrane proteins that are useful for serodiagnosis (45, 62, 63, 68). Our alignment results showed a distinct fragment (∼20 amino acids) in A. platys Omp-1X that was not observed in the closest homologs from Anaplasma and Ehrlichia spp. Furthermore, this region was identical in A. platys samples from the geographically separated regions of Venezuela and Taiwan. This specific OMP-1X peptide antigen did not cross-react with anti-A. phagocytophilum serum and, therefore, may be suitable for species-specific differential serodiagnosis of A. platys.

We employed touchdown PCR in the present study because the only available source of A. platys DNA was a small amount of DNA purified from the blood of naturally infected dogs. Incorrect base calls during the amplification were minimized by using high-fidelity Taq polymerase. Only a few A. platys gene sequences have previously been reported, including the 16S rRNA, groEL, and gltA genes (3, 33, 67). Application of the molecular approach used here should facilitate the identification of additional DNA sequences to further our understanding of the A. platys genome.